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利用减毒沙门氏菌为载体将4-1BBL体内转染抗原提呈细胞,观察体内4-1BBL基因转染对大鼠免疫功能的影响。构建含pIRES2-EGFP-4-1BBL质粒的减毒沙门氏菌(疫苗菌),通过体外感染HepG2细胞观察疫苗菌的侵袭功能和携带外源基因能力,验证疫苗菌作为4-1BBL基因转染载体的作用。疫苗菌2周内连续灌服大鼠3次,末次灌服后14 d心脏取血处死,RT-PCR和荧光显微镜检测报告绿色荧光蛋白(GFP)在脾脏细胞中的转录表达,流式细胞仪检测外周血和脾脏中细胞表型变化。疫苗菌在体外能成功的将外源基因携带至HepG2细胞进行表达,灌服疫苗菌的大鼠外周血和脾脏细胞4-1BBL和4-1BB表达明显升高,外周血中CD3+、CD3+CD8+和CD3+CD25+,脾脏细胞中CD3+CD8+、CD3+CD25+也有明显的增高。表明含4-1BBL基因的减毒沙门氏疫苗菌能实现体内基因转染,4-1BBL的高表达能增强机体的细胞免疫功能。
Using attenuated Salmonella as the carrier, the 4-1BBL antigen was transfected into the cells in vivo to observe the effect of 4-1BBL gene transfection in vivo on immune function in rats. To construct an attenuated Salmonella typhimurium (vaccine strain) containing pIRES2-EGFP-4-1BBL plasmid and infect HepG2 cells in vitro to observe the invasive function and foreign gene-carrying ability of the vaccine strain, and to verify the role of the vaccine strain as a 4-1BBL gene transfection vector . The vaccine was administered 3 times a day for 2 weeks, and the heart was sacrificed on the 14th day after the last oral administration. Transcriptional expression of green fluorescent protein (GFP) in spleen cells was detected by RT-PCR and fluorescence microscopy. Flow cytometry Detection of cell phenotypes in peripheral blood and spleen. In vitro, the vaccine strains could successfully carry exogenous gene to HepG2 cells for expression. The expression of 4-1BBL and 4-1BB in peripheral blood and spleen cells of the vaccine-vaccinated rats were significantly increased. The levels of CD3 +, CD3 + CD8 + And CD3 + CD25 +, spleen cells in CD3 + CD8 +, CD3 + CD25 + also significantly increased. It is indicated that the attenuated Salmonella typhimurium vaccine strain containing 4-1BBL gene can achieve gene transfection in vivo. The high expression of 4-1BBL can enhance the cellular immune function of the body.